1. Field of the Invention
The present invention relates to a method for preventing and/or treating osteoporosis and other disease states in animal subjects, utilizing organic boron compounds.
2. Description of the Related Art
The adult skeleton is composed of 80% cortical and 20% trabecular bone. Bone matrix is composed of 25% collagen, 65% inorganio material, and 10% non-cellular proteins (i.e., osteocalcin, silaoprotein, proteoglycans and osteonectin) and lipids. Osteoblasts synthesize and secrete Type 1 collagen and mucopolysaccharides to form the bone matrix which is laid down between the thin layers of osteoid. The layers are subsequently mineralized with 99% of the body's calcium found in the bone as a calcium phosphate complex with hydroxyapatite. Osteoblasts also synthesize and release Pg-E.sub.2, osteocalcin, osteonectin, and collagenase. These cells have membrane receptors for PTH, VitD, and glucocorticosteroids.
Osteoclasts are multi-nucleated cells which reabsorb calcium from bone and cartilage. These cells may be derived from hemopoietic stem cells, i.e., mono-nucleated phagocytic cells. These cells lie on the bone surface (Howship's lacunae) and the surface becomes ruffled (motile microvilli). This area is sealed off from the neighboring cells. In these pockets, acid is produced which activates acidic hydrolytic enzymes and neutral proteases are released. Calcium is chelated by organic anions, e.g., citrate.
Osteoblasts release collagenase which removes protein from the surface of the bone, thus allowing the osteoclasts to attach. Collagenase cleaves the native helical molecule of collagen. The other proteolytic enzymes, e.g. neutral and acidic cathepsins, digest the protein further with the release of hydroxyproline to the extracellular compartment, which is an index of bone resorption. Hydroxyapatite protects collagen from denaturation. Collagenase and stromelysin are extracellular calcium dependent, zinc endoproteinases which hydrolyze collagen. Proteoglycan is thought to be a substrate for stromelysin (matrixin, proteoglycanase, transin, and MMP-3). These enzymes exist as zymogens or proenzymes. The proenzymes are activated by organomercurials, serine proteinases, oxidants and surfactants. The amino acid sequences of the metalloproteinases are known from the corresponding cDNA. The activity of the enzyme is inhibited by endogenous tissue inhibitors (TIMP). In humans a collagenase inhibitor has been identified in the alpha-2 macroglobulin fraction of plasma. Previous studies have shown that hydroxamates, thiol analogs, phosphorous-based derivatives, eriochrome, black T and gold inhibit collagenase, HFC activity.
Osteoporosis is generally associated with a reduced trabecular bone volume leading to increased risk of bone fractures. This process is probably due to a metabolic imbalance between the rates of new bone formation and bone resorption. Osteoporosis can be divided into two classes: (1) type I or post-menopausal,which is related to reductions in estrogen content and affects primarily trabecular bone, and (2) type II or senile, which is related to reduced calcium absorption and affects primarily cortical bone.
Bone resorption can be divided into two processes which are probably being carried out concurrently. Phase I involves the inorganic metabolism conducted principally by osteoclasts, macrophages, monocytes, PMNs, and fibroblasts. This process is regulated by PTH, Pg-E.sub.2, and cAMP which activate lysosomal hydrolytic enzymes and causes solubilization of the minerals in the bone, releasing calcium to the blood. Phase II involves organic metabolism where there is proteolytic destruction of the bone matrix collagen, releasing hydroxyproline to the blood. This process is initiated by the release of collagenase and cathepsin D from osteoblasts at the bone surface. The cellula enzymes belong to the metalloproteinase group of proteolytic enzymes which usually function at neutral pH. PTH binds to membrane receptors on osteoblasts, pre-osteoblasts and osteocytes, which activates the release of calcium from the dense bone, probably due to the activation of lysosomal enzymes, e.g., cathepsins, cAMP, interleukin-1, or prostaglandins.
In the elderly population there is a decrease of estrogen, progesterone, testosterone and vitamin D3 levels with age. With advanced age there is a reduction of calcitonin which severely reduces calcium absorption from the gut. There is a positive correlation between the extracellular reduction of these physiological parameters and with osteoporosis. Other factors are: smoking, lack of exercise, sunlight, and disease states like myeloma, skeletal metastasis, gastric surgery, anti-convulsant therapy, male hypogonadism, thyrotoxicosis, amenorrhea, anorexia nervosa, hyperprolactinanemia, diabetes mellitus, immobilization, osteogenic imperfecta, and homocystinuria.
A number of agents have been noted to attenuate loss of bone mass in elderly humans or to accelerate bone growth in the young, such as estrogens, insulin, fluorides, anabolic steroids, calcitonin, growth hormone, fibroblast growth factor, transforming growth factor, epidermoid growth factor, bone morphogenic protein (osteogenin), diphosphates, and oral calcium supplements, with varying degrees of success. Most evidence today indicates that massive intakes of calcium (1500-2000 mg/day) orally does not prevent bone loss in post-menopausal women. It is not clear what the mechanism of action of estrogen is in blocking bone resorption. Estrogen therapy increases calcitonin in menopausal women which activates the metabolite of Vit-D3 for calcium absorption from the gut. There may be more receptors in the gut for the absorption of calcium in the presence of estrogen and there is an increase in calcium binding protein. Progesterone probably facilitates calcium flux into bone. Calcitonin acts directly on osteoclasts via cAMP to inhibit bone resorption.
Bone mass is decreased by treatment with the following drugs over a long period of time: glucocorticoids, thyroxine, heparin, cytotoxic drugs, retinoids [vit A], phorbol esters, Pg-E's, interleukin-1, endotoxins and parathyroid hormone (PTH). Apparently PTH stimulates production and relase of cAMP and prostaglandin synthesis which is coupled with the release of collagenase. This causes the osteoblasts to become flattened and fibroblastic in shape. They release chemical substances, Pg-E.sub.2, which attract osteoclasts to the surface of the bone. Pg-E.sub.2 triggers the release of osteoclasts activating factor. At the surface there is local production of acids and carbonic anhydrase II, an isoenzyme. Inhibition of these metabolic processes also slows the bone resorption process.
At this stage there is an influx of other types of cells, e.g., macrophages, fibroblasts and monocytes which can continue the resorption process. Certain mechanisms in the movement of calcium from blood to bone are known. The metabolite of vitamin D, 1,25(OH)2Vit-D3, induces calcium transport by binding to a specific cytosol/nuclear receptor and forming a complex. This complex binds to chromatin DNA increasing template activity and initiating the synthesis of the calcium binding protein. Corticosteroids are known to suppress prostaglandin synthesis and reduce the levels of osteocalcin (Gla protein, or BGP). This protein stimulates osteoblastic activity.
There are data which suggest that corticosteroids modulate the number of receptors on osteoblasts for 1,25(OH)2Vit-D3, as well as alkaline phosphatase and collagen synthesis. Sodium fluoride stimulates osteoblast function by recruiting an increased number of osteoblasts. There is evidence that fluorapatite crystals form which are more resistant to resorption. Bisphosphonates are inhibitors of crystallization and calciferication of bone. Heparin increases the production of collagenase and enhances its release and activity. Thus there are numerous sites within the regulation of calcium metabolism and proteolytic enzymes where therapeutic agents may function.
Bone resorption appears to be blocked by sulfanilamide, acetazolamide, methazolamide, benzolamide, ethoxzolamide, flurbiprofen and boron. No doubt a number of these agents are inhibiting the carbonic anhydrase enzyme so that hydrogen ions are not generated to produce an acidic medium. Other agents like thiazides inhibit calcium excretion in the urine. Boron depletion depresses growth in chicks when inadequate amounts of cholecalciferol is in the diet. Boron treatment (3.23 mg/day) of postmenopausal women has been shown to result in higher plasma levels of calcium and serum 25-hydroxycholecalciferol and lower levels of calcitonin and osteocalcin. Recent work at the U.S. Department of Agriculture (Grand Forks, N. Dak.) has shown that inorganic boron, e.g., boric acid or sodium borate, in the diet at 3 mg/day blocks the urinary excretion of calcium, magnesium and perhaps phosphorus. Boron supplements markedly elevate estrogen and testosterone levels and supposedly block the process of osteoporosis. Dietary boron is useful in cholecalciferol-deficient chicks to correct cartilage growth.
Unfortunately, the intake of boron in the form of boric acid, or boric acid salts, has a number of undesirable side effects. The intake of boric acid (at dosages of 250 mg/kg for 16 days) has been found to cause severe degeneration of the testes with giant cell formation, pyknosis and exfoliation. Further, such intake of boric acid causes an increase in alkaline phosphatases in tissues, hepatic glycogen content, and maintenance of body fat. Nausea, vomiting, diarrhea, dermatitis, depressed growth, and lethargy occur in adults treated with boron as a trace element, in inorganic forms. Further, the metabolization of boron from such norganic boron compounds is relatively inefficient. Finally, the marked elevation in estrogen and testosterone levels is associated with undesirable side effects in some instances, including mood disorders, water retention, Na.sup.+ retention, thromboembolism, endometrial hyperplasma, cholestatic jaundice, headaches, gastrointestional distress, virilization, and carcinoma of the biliary tract.
Accordingly, it would be a significant advance in the art to provide a method of combatting osteoporosis, which is not subject to the aforementioned deficiencies associated with boric acid and boric acid salts.
It would be particularly desirable to provide a method of combatting osteoporosis, which is bioactive via a non-estrogenic mechanism.
Accordingly, it is an object of the present invention to provide an improved method of combatting osteoporosis, which is free of the aforementioned deficiencies.
It is another object of the present invention to provide a method of combatting osteoporosis utilizing boron in a form taken up by cells more effectively than boric acid or borate salts, and which combats osteoporosis by different mechanisms than the boric acid and boric acid salt compounds used for such purpose in the prior art.
Other objects and advantages of the present invention will be more fully apparent from the ensuing disclosure and appended claims.